A conserved phosphorylation switch controls the interaction between cadherin and β-catenin in vitro and in vivo

Abstract

In metazoan adherens junctions, β-catenin links the cytoplasmic tail of classical cadherins to the F-actin-binding protein α-catenin. Phosphorylation of a Ser/Thr-rich region in the cadherin tail dramatically enhances affinity for β-catenin and promotes cell-cell adhesion in cell culture systems, but its importance has not been demonstrated in vivo. Here, we identify a critical phosphorylated serine in the C. elegans cadherin HMR-1 required for strong binding to the β-catenin homolog HMP-2. Ablation of this phosphoserine interaction produces developmental defects that resemble full loss-of-function (Hammerhead and Humpback) phenotypes. Most metazoans possess a single gene for β-catenin, which is also a transcriptional coactivator in Wnt signaling. Nematodes and planaria, however, have a set of paralogous β-catenins; for example, C. elegans HMP-2 functions only in cell-cell adhesion, whereas SYS-1 mediates transcriptional activation through interactions with POP-1/Tcf. Our structural data define critical sequence differences responsible for the unique ligand specificities of these two proteins.

Figure 1. HMP-2 structure and dependence of binding on the phosphorylation state of HMR-1

(A) Four constructs of HMP-2, which were used in our study, are shown and their residue boundaries indicated. On the right side of each construct, K_D values for pHMR-1_cyto80 and HMR-1_cyto80, measured by ITC experiments are shown. HMP-2_13end and HMP-2_54end did not give measurable ITC signals for the interaction with HMR-1_cyto80 and are labeled as ND. (B) Structure of HMP-2_54arm (residues 77–613). Helices 1 and 2 of each arm repeat are colored yellow, and helix 3 is colored light green. The structures of HMP-2_54arm and HMP-2_54end are very similar except for the N-terminal region. Residues 56–79 are seen only in the HMP-2_54end structure, and form an extra N-terminal helix whose position appears to be a consequence of crystal packing. (C) Superposition of arm repeats 1 to 4 of HMP-2 and mouse β-catenin (PDB ID 1I7W). HMP-2 helices are colored as in Figure 2A and β-catenin is colored grey. (D) Electrostatic surface of the HMP-2 arm domain, with negative and positive regions colored red and blue, respectively. Contoured at ± 5 k_BT/e.

Figure 2. Sequence alignment of the cytoplasmic tails of mouse E-cadherin and HMR-1 reveals key conserved residues

Juxtamembrane region including p120 (JAC-1 in C. elegans) binding site was aligned base on the sequence homology and β-catenin (HMP-2) binding region was aligned based on the crystal structures of pE_cyto and pHMR-1_cyto80. HMP-2 binding domain is divided into four regions and each region is indicated with magenta bar on top of the sequences. The C-terminal cap region is only present in E_cyto and is shown with grey colored bar. The thickened rectangles represent α-helices. Five CKI-phosphorylated sites in HMR-1_cyto are marked with triangle and six phosphorylation sites by GSK-3β and CKII in E_cyto are circled. Phosphorylated residues observed in the structures are written in bold (3 residues in pE_cyto and 4 in pHMR_cyto80). The starting residue numbers are indicated.

Figure 3. Crystal structure of HMP-2_54arm/pHMR-1_cyto80 complex identifies key interaction regions

In each panel, the arm domain colored as in Figure 1B and the structures of pHMR-1_cyto80 are shown in salmon (conformation A from the high-resolution P4₃ crystal form) and magenta (conformation B). (A) Ribbon diagram of the complex. The N and C terminus of each protein is labeled. Phosphorylated E_cyto is aligned to pHMR-1_cyto80 and shown in grey. The four regions of pHMR-1 are boxed and labeled as I, II, III and IV. (B) Interaction region I. Arm repeats 7, 8 and 9 are labeled as R7, R8 and R9. Side chains of amino acids that make direct contacts between HMP-2 and pHMR-1_cyto are shown as sticks, and HMP-2 and pHMR-1 residues are labeled in black and magenta, respectively. Hydrogen bonds are shown as dotted lines. (C) Interaction region II. (D) Interaction region III. Most of the contacts between HMP-2 and pHMR-1_cyto are formed by side chains, except for N317, N356, and H400 of HMP-2, which form hydrogen bonds with amide and carbonyl groups of a polypeptide backbone of pHMR-1_cyto.

Figure 4. Phosphorylation generates specific interactions of HMR-1 region IV with HMP-2

(A) Structures of HMP-2 bound to the two different conformations of pHMR-1_cyto are aligned, with conformations A and B shown in salmon and magenta. Also shown is a superposition of pE_cyto (grey) from the β-catenin_arm/pE_cyto complex structure (PDB ID 1I7W) with conformation B of pHMR-1_cyto; E-cadherin residues are indicated in parentheses. For clarity, the superimposed arm domain β-catenin is not shown. All phosphorylated residues observed in the structures of pE_cyto and pHMR-1_cyto are represented. (B) Close-up of the interactions between HMP-2 and pS1212 of pHMR-1_cyto from conformations A and B. (C) Comparison of HMR-1 pS1212 interactions with HMP-2 and E-cadherin pS686 with β-catenin. (D) Comparison of β-catenin–pE_cyto interactions with those of HMP-2–pHMR-1_cyto residues 1214–1219.

Figure 5. HMR-1 pS1212 is required for interaction between HMP-2 and HMR-1

(A, B) DIC (A) and confocal (B) images of 1.5-fold elongating hmr-1(zu389) homozygotes rescued to viability by HMR-1::GFP. Lethality in offspring of hmr-1(zu389); hmr-1::gfp mothers is 85.2% (n = 209). Signal localizes to epidermal adherens junctions similarly to HMR-1 immunostaining (not shown). Scale bar is 10 µm. (C, D) The phospho-null construct HMR-1(S1212A)::GFP localizes to junctions but is unable to rescue hmr-1(zu389) homozygotes to viability. Lethality in offspring of hmr-1(zu389)/+; hmr-1(S1212A)::gfp mothers is 27.4% (n = 1072). Cells in (D) appear compressed due to retraction of the hypodermis subsequent to failure of epiboly. (E, F) HMR-1(T1215A, S1218A)::GFP localizes to junctions and rescues hmr-1(zu389) embryonic enclosure and elongation. The hmr-1 transgene reduces embryonic lethality of hmr-1(zu389)/+ offspring from 24.9% (n=1101) to 16.9% (n=1055, 3 independent lines). Pictured is a representative offspring from a hmr-1(zu389)/+; hmr-1(T1215A, S1218A)::gfp mother. (G, H) Wildtype HMP-2::GFP localizes to junctions (H) and rescues hmp-2(zu364) to viability (G). (I) hmp-2(zu364) homozygotes fail to elongate and die with the Humpback phenotype. The zu364 lesion was identified as a point mutation resulting in HMP-2 R271C. (J) HMP-2(R271C)::GFP localizes exclusively to the cytoplasm, and embryos die with the Hmp phenotype, consistent with a model in which this residue is crucial for interacting with HMR-1 pS1212. Arrowhead indicates dorsal humps.

Fig 6. HMP-2 Y599E localizes aberrantly and produces mild circumferential F-actin bundle (CFB) defects

(A,B) Wildtype HMP-2::GFP signal is well established by early elongation (A) and is maintained at junctions through late elongation (B). Rescued zu364 homozygotes display 53.9% lethality (n = 178), approximately commensurate with the transmission rate of the extrachromosomal array carrying the transgene. Scale bar is 10 µm. (C,D) HMP-2(Y599F)::GFP rescues hmp-2(zu364) homozygotes and localizes to epidermal adherens junctions identically to wildtype HMP-2::GFP both in early (C) and late (D) elongation. Rescued zu364 homozygotes display 50.2% lethality (n = 305). (E,F) HMP-2::GFP Y599E rescues hmp-2(zu364). Signal initially localizes to adherens junctions (E), but it becomes punctate during late elongation, and fluorescent excursions form orthogonally to junctions between lateral (seam) cells and their dorsal and ventral neighbors (F). Rescued zu364 homozygotes display 45.9% lethality (n = 283). (G–I) CFBs visualized by phalloidin staining in hmp-2(zu364) homozygotes rescued by wildtype (I) or Y599E (G, H) HMP-2::GFP constructs. Wildtype HMP-2::GFP embryos display evenly-distributed radial F-actin bundles (I). CFBs in Y599E embryos become irregularly spaced (G, H), and occasionally multiple bundles aggregate to a single locus on the junction (H, arrowhead). Scale bar is 10 µm.

Figure 7. Structural comparison of HMP-2/pHMR-1_cyto80 complex with SYS-1/POP-1 complex suggests why only HMP-2 can bind pHMR-1

(A) Superposition of SYS-1/POP-1 (PDB ID 3C2G) into HMP-2/pHMR-1_cyto80 complex. SYS-1 and POP-1 are colored light blue and cyan, respectively. Repeats 7 to 9 of SYS-1 and POP-1 are manually aligned to corresponding regions of HMP-2 and pHMR-1 in Coot. POP-1 (amino acids 8–14) aligns well with pHMR-1 region III, as shown in the close-up (boxed) view. SYS-1 and POP-1 residue numbers are in parentheses, and only those SYS-1 side chains that interact with POP-1 interaction are shown. Side chain of Glu9 of POP-1 is not modeled in the original structure. Hydrogen bonds in HMP-2/pHMR-1_cyto80 and SYS-1/POP-1 complexes are shown as black and blue dotted lines, respectively. (B) Repeats 5 and 6 of SYS-1 are aligned to corresponding region of HMP-2. Three arginine residues of HMP-2 interacting with pS1212 are not conserved in SYS-1.